Cu-dependent APP phosphorylation drives its trafficking Phosphorylation of Amyloid Precursor Protein at Threonine-668 is Essential for its Copper- Responsive trafficking in SH-SY5Y neuroblastoma cells

Amyloid Precursor protein (APP) undergoes post-translational modification, including Oand N-glycosylation, ubiquitination and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at Threonine 668 (T668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phosphositespecific antibody) exhibited a copperdependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, T668 to Alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the T668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at T668 in neurons. Our results show that the GSK3-β kinase inhibitors LiCl, SB 216763 and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3-β dependent phosphorylation in SHSY5Y cells. 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M113.538710 The latest version is at JBC Papers in Press. Published on March 7, 2014 as Manuscript M113.538710 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on A uust 1, 2016 hp://w w w .jb.org/ D ow nladed from Cu-dependent APP phosphorylation drives its trafficking Amyloid Precursor Protein (APP) is an integral type I trans-membrane protein that is synthesized in the endoplasmic reticulum and transported through the Golgi network via the secretory pathway where it undergoes post-translational modifications including Nand Oglycosylation, ubiquitination and phosphorylation (1-11). A proportion of APP reaches the plasma membrane where it is rapidly endocytosed and trafficked through endocytic and recycling compartments back to the cell surface or degraded by lysosomes (reviewed in (12,13)). The trafficking process of APP has been intensively studied as it is closely linked to its processing and the generation of the toxic amyloid β peptide (Aβ) peptide central to Alzheimer’s disease (AD) pathogenesis. APP can be cleaved by α, β and γsecretases, which are localized to specific subcellular compartments. For instance, APP is cleaved by the β-secretase BACE1 in acidic compartments (the trans-Golgi, early endosomes) to generate a soluble ectodomain (sAPPβ) and a C-terminal fragment (β-CTF). C-terminal fragments can be further processed by the γ-secretase complex, which resides in the endocytic compartment or late endosomes to release the Aβ peptide. Processing by βand γsecretases to generate Aβ is referred to as the amyloidogenic processing pathway (reviewed in (12,13)). α-secretase cleavage (non-amyloidogenic) occurs primarily at the plasma membrane (PM). There has been much debate as to the normal function of APP. In neurons APP function has been associated with neurite outgrowth, neuronal migration and repair via interaction with extracellular matrix proteins (14-16). APP undergoes rapid kinesin-1 dependent anterograde transport and reaches presynaptic terminals (17,18). At the synapse, APP is involved in synapse formation, synaptic transmission, plasticity and learning and memory (reviewed in (19)). Relevant to this study, APP is also involved in copper homeostasis (20,21). Identifying the cellular signals, which mediate APP trafficking is central to understanding its normal function and processing, including yielding the toxic Aβ peptide of Alzheimer’s disease. The subcellular localization of APP is regulated by phosphorylation at a number of sites within the intracellular domain. APP is phosphorylated at eight residues (Y653, T654, S655, S675, T668, Y682, T686 and Y687; APP695 numbering), within the APP intracellular domain (AICD). Phosphorylation at these sites has been reported to impact APP processing and cellular localization (reviewed in (22)). The phosphorylation of APP at Threonine-668 (T668) results in a significant conformational change that may affect interactions with binding partners and hence impact its subcellular localization and metabolism (23). Phosphorylation at T668 is a normal process associated with neurite extension, anterograde transport of vesicular cargo and in signaling to the nucleus (24-28). APP is phosphorylated at T668 in vitro and in vivo by a number of kinases including glycogen synthase kinase 3-β (GSK-3β), Jun N-terminal kinase-3 (JNK3), cell division cycle protein (Cdc2) and Cyclin-dependent kinase 5 (Cdk5) (7,29-32). Whether and how phosphorylation at T668 impacts APP processing remains controversial with studies showing varying results. For instance, one study reported that phosphorylation of APP at T668 increased Aβ production by enhancing β-secretase cleavage (33) whilst a later study showed a decrease in Aβ due to the inhibition of γ-secretase cleavage (34). In contrast, knock-in mice expressing APP with a threonine to alanine substitution showed no change in APP metabolism including brain levels of Aβ (35). A recent study has shown that non-phosphorylated forms (at T668) of C-terminal APP fragments are associated with lipid raft-like micro-domains where the γsecretase complex (amyloidogenic) resides, whereas T668 phosphorylated C-terminal fragments reside pre-dominantly in cytoplasmic fractions (36). Hence phosphorylation regulates the localization of APP and thus affects its processing by γsecretases (36). We have previously reported that copper promotes the re-localization of APP from a predominant Golgi localization to a wider distribution (37) including the PM, 2 by gest on A uust 1, 2016 hp://w w w .jb.org/ D ow nladed from Cu-dependent APP phosphorylation drives its trafficking which is the predominant site of nonamyloidogenic cleavage by α-secretase. Copper-responsive APP trafficking was due to both a stimulation of exocytosis and suppression of endocytosis of APP (37). Our earlier studies on the copper transport protein which is mutated in Menkes disease, ATP7A, demonstrated that copper induces the trafficking of ATP7A via phosphorylation at specific residues in its C-terminus (38). This was demonstrated by targeted mutagenesis of phosphorylatable residues. In the current study we investigated whether phosphorylation at T668, a widely studied phosphorylation site, is required for copper-responsive APP trafficking. We investigated this by (1) studying copper-responsive trafficking of a phospho-deficient mutant T668A (2) studying the level of phosphorylated T668 using a phosphosite-specific antibody following copper treatment and (3) using kinase inhibitors including lithium chloride (LiCl) to inhibit phosphorylation at T668. Our results from these various approaches strongly suggest that copper promotes a re-localization of APP by phosphorylation at T668 in the neuronal cell model SH-SY5Y. This involves GSK3-β and importantly identifies a novel mechanism by which copper can regulate APP function in neuronal cells. EXPERIMENTAL PROCEDURES Antibodies and ReagentsThe following antibodies were used in this study: GM130 (BD Transduction Laboratories), β-catenin (abcam), Ankyrin-G (NeuroMab, Davis, CA), C20 (C-terminal APP antibody; calbiochem), Phospho-APP (T668 (D90B8); Cell Signaling Technology); β-actin (Sigma) and W0-2. The antibody CT77 was used to detect the copper transport protein, ATP7A, and was a kind gift from Prof.B. Eipper (Neuroscience and Molecular, Microbial and Structural Biology Division, University of Connecticut). GM130, and Ankyrin-G were used as markers for the cis-Golgi network and as an axonal marker in primary hippocampal neurons, respectively. The C-terminal APP antibody C20 specifically recognizes residues 751-770 and will detect full-length APP and C-terminal fragments. The W0-2 epitope lies within the Aβ domain (1-4 amino acids) and will detect full-length APP as well as the sAPP-α ectodomain and Aβ peptide. Lithium Chloride (Sigma) was used as a GSK3-β inhibitor. Other kinase inhibitors for GSK3-β and cyclin-dependent kinases were obtained from the Tocriscreen Kinase Inhibitor Toolbox (Tocris Bioscience). PhosSTOP Phosphatase inhibitor cocktail tablets (Roche) were used to inhibit phosphatase activity following cell lysis. Western lysis buffer was also supplemented with Complete EDTA-free protease inhibitor cocktail tables (Roche). Cell culture and generation of stable cell linesHuman neuroblastoma SH-SY5Y cells (American Type Culture Collection catalog no. CRL-2266) were cultured in DMEM (Invitrogen) containing GLUTAMAX-I (Invitrogen) supplemented with 10% fetal calf serum and 1 mM sodium pyruvate. Cell lines were cultured at 37oC and in the presence of 5% CO2. To generate SHSY5Y stable cell lines, cells grown in 6-well plates were transfected with 2.4 μg of plasmid DNA using the Lipofectamine 2000 reagent (Invitrogen) according to manufacture’s instructions. Stable SH-SY5Y cell lines were selected and maintained with Geneticin (0.5 mg/ml; Invitrogen) 48 h following transfections. The SH-SY5Y cell lines generated express APP695 or APP with point mutations at the threonine 668 or the serine 655 residue with a C-terminal mCherry fluorescent tag in the pcDNA3.1 vector (Invitrog